Increased extracellular matrix (ECM) deposition and airway smooth muscle (ASM) mass are major contributors to airway remodeling in asthma. Recently, we demonstrated that the ECM protein collagen I, which is increased surrounding asthmatic ASM, induces a proliferative, hypocontractile ASM phenotype. Little is known, however, about the signaling pathways involved. Using bovine tracheal smooth muscle, we investigated the role of focal adhesion kinase (FAK) and downstream signaling pathways in collagen I–induced ASM phenotype modulation. Phosphorylation of FAK was increased during adhesion to both uncoated and collagen I–coated culture dishes, without differences between these matrices. Nor were any differences found in cellular adhesion. Inhibition of FAK activity by overexpression of the FAK deletion mutants FAT (focal adhesion targeting domain) and FRNK (FAK-related nonkinase) attenuated adhesion. After attachment, FAK phosphorylation increased in a time-dependent manner in cells cultured on collagen I, whereas no activation was found on an uncoated plastic matrix. In addition, collagen I increased in a time- and concentration-dependent manner the cell proliferation, which was fully inhibited by FAT and FRNK. Similarly, the specific pharmacologic FAK inhibitor PF-573228 [6-((4-((3-(methanesulfonyl)benzyl)amino)-5-trifluoromethylpyrimidin-2-yl) amino)-3,4-dihydro-1H-quinolin-2-one] as well as specific inhibitors of p38 mitogen-activated protein kinase (MAPK) and Src also fully inhibited collagen I–induced proliferation, whereas partial inhibition was observed by inhibition of phosphatidylinositol-3-kinase (PI3-kinase) and mitogen-activated protein kinase kinase (MEK). The inhibition of cell proliferation by these inhibitors was associated with attenuation of the collagen I–induced hypocontractility. Collectively, the results indicate that induction of a proliferative, hypocontractile ASM phenotype by collagen I is mediated by FAK and downstream signaling pathways.
Airway hyperresponsiveness, persistent airway obstruction, and decline in lung function are characteristic features of chronic asthma (Bousquet et al., 2000). Airway remodeling, including increased airway smooth muscle (ASM) mass and altered extracellular matrix (ECM) deposition, is considered to contribute to these features (Bousquet et al., 2000; Jeffery, 2001; Dekkers et al., 2009a). Increased ASM mass may comprise hyperplasia as well as hypertrophy (Ebina et al., 1993), and in keeping with hyperplasia ASM cells display phenotype plasticity and may reenter the cell cycle (Halayko et al., 2008). Thus, exposure of ASM cells to mitogenic stimuli results in the induction of a proliferative phenotype associated with a decreased contractile function (Hirst et al., 2000b; Gosens et al., 2002; Dekkers et al., 2007, 2012). Phenotype plasticity is a dynamic and reversible process, and removal of mitogenic stimuli, for example, by serum deprivation in the presence of insulin, results in the reintroduction of a (hyper)contractile ASM phenotype (Ma et al., 1998; Schaafsma et al., 2007; Dekkers et al., 2009b).
From biopsy studies, it has become apparent that ECM deposition is increased beneath the epithelial basement membrane in the airways of asthmatics (Roche et al., 1989). In addition, the total amount of ECM in the microenvironment of the ASM is increased as well (Bai et al., 2000) and may involve deposition of various ECM proteins, including collagen type I (Roberts and Burke, 1998). The ECM in the ASM layer plays a key role in determining its physical and mechanical properties. In addition, ECM proteins may affect ASM phenotype switching. Growth factor-induced phenotype switching of ASM cells is inhibited by culturing the cells on laminin-111, resulting in the maintenance of a contractile ASM phenotype (Hirst et al., 2000a; Dekkers et al., 2007, 2010). Conversely, culturing of ASM cells on monomeric collagen type I enhances growth factor-induced proliferation (Hirst et al., 2000a; Nguyen et al., 2005; Dekkers et al., 2010). Moreover, collagen I induces a hypocontractile, proliferative ASM phenotype in intact human and bovine tracheal smooth muscle preparations in the absence of other mitogens (Dekkers et al., 2007, 2012). ASM cells obtained from asthmatics produce more collagen I compared with cells obtained from healthy subjects (Johnson et al., 2004). In addition, nonasthmatic ASM cells cultured on an ECM laid down by asthmatic ASM cells proliferate more rapidly and vice versa (Johnson et al., 2004), suggesting that changes in the ECM profile may contribute to enhanced asthmatic ASM growth in situ.
Integrins are a group of heterodimeric transmembrane glycoproteins linking the ECM to the intracellular compartment (Giancotti and Ruoslahti, 1999). The collagen-binding integrin α2β1 is the main integrin involved in collagen I–induced ASM cell attachment, ASM cell proliferation, cytokine production, and glucocorticosteroid resistance (Nguyen et al., 2005; Fernandes et al., 2006). In addition, the fibronectin-binding integrins α4β1 and α5β1 appeared important in the enhancement of platelet-derived growth factor (PDGF)–induced proliferation by collagen I, whereas the fibronectin-binding integrin αvβ3 was also required for attachment to collagen I (Nguyen et al., 2005). Recently, we demonstrated that the α5β1 integrin is also of major importance in collagen I–induced increase of basal proliferation (Dekkers et al., 2010). No information is yet available on the signaling pathways of ECM-integrin interactions in ASM cells. From other cell types it is known that most integrins activate focal adhesion kinase (FAK), resulting in autophosphorylation at Tyr397 and generating a binding site for Src, which then phosphorylates a number of other tyrosine residues on FAK (Giancotti and Ruoslahti, 1999; Hynes, 2002; Cox et al., 2006). FAK may subsequently activate downstream signaling cascades, including the phosphatidylinositol-3-kinase (PI3-kinase) and mitogen activated protein kinase (MAPK) pathways (Giancotti and Ruoslahti, 1999)
We explored the role of FAK and downstream signaling pathways in collagen I–induced ASM phenotype switching. Using bovine tracheal smooth muscle (BTSM) cells, we examined the effects of monomeric collagen I on FAK phosphorylation during adhesion and proliferation. The role of FAK in these processes was assessed by overexpression of FAK and of the FAK deletion mutants FAT [derived from the focal adhesion targeting (FAT) domain of FAK] and FRNK (FAK-related nonkinase), which inhibit FAK localization to the focal adhesions and FAK activation (Hildebrand et al., 1993; Richardson and Parsons, 1996). In addition, by pharmacologic inhibition of FAK, Src, mitogen-activated protein kinase kinase (MEK), PI3-kinase, and p38 MAPK, we investigated the contribution of these pathways to collagen I–induced BTSM proliferation and hypocontractility.
Materials and Methods
Tissue Preparation and Organ-Culture Procedure.
Bovine tracheae were obtained from local slaughterhouses, and BTSM strips of macroscopically identical length (1 cm) and width (2 mm) were prepared as described elsewhere (Dekkers et al., 2007). Muscle strips were washed in Medium Zero [sterile Dulbecco’s modified Eagle’s medium (DMEM), supplemented with sodium pyruvate (1 mM), nonessential amino-acid mixture (1:100), gentamicin (45 µg/ml), penicillin (100 U/ml), streptomycin (100 µg/ml), amphotericin B (1.5 µg/ml), apo-transferrin (5 µg/ml, human), and ascorbic acid (0.1 mM)] and transferred into suspension culture flasks. The strips were maintained in culture in Medium Zero using an Innova 4000 incubator shaker (37°C, 55 rpm) for 4 days. When applied, monomeric collagen type I (50 µg/ml), PF-573228 (FAK inhibitor II, 100 nM) [6-((4-((3-(methanesulfonyl)benzyl)amino)-5-trifluoromethylpyrimidin-2-yl) amino)-3,4-dihydro-1H-quinolin-2-one], PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-day]pyrimidine] (10 μM), U0126 (1,4-diamino-2,3-dicyano-1,4-bis [2-aminophenylthio]butadiene) (3 μM), LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] (10 μM), and/or SB203580 [(4-[5-(4-fluorophenyl)-2-[4-methylsulphonyl)phenyl]-1H-imidazol-4-yl]pyridine) (10 μM) were present during the entire incubation period.
Isometric Tension Measurements.
Isometric tension measurements were performed as described elsewhere (Dekkers et al., 2007). Briefly, BTSM strips were washed with Krebs-Henseleit buffer [composition (mM): NaCl 117.5, KCl 5.60, MgSO4 1.18, CaCl2 2.50, NaH2PO4 1.28, NaHCO3 25.00 and glucose 5.50, pregassed with 5% CO2 and 95% O2; pH 7.4 at 37°C]. Subsequently, the strips were mounted for isometric recording in organ baths. During a 90-minute equilibration period, the resting tension was gradually adjusted to 3 g. Subsequently, the BTSM strips were precontracted with 20 and 40 mM KCl solutions. Collectively, the total washout period before the start of the isometric tension experiments was at least 3 hours. After washing, maximal relaxation was established by the addition of (−)-isoproterenol (0.1 μM; Sigma-Aldrich, St. Louis, MO). The tension was readjusted to 3 g, and after another equilibration period of 30 minutes the cumulative concentration response curves were constructed to methacholine. When maximal tension was reached, the strips were washed, and maximal relaxation was established using isoproterenol (10 μM).
Isolation of Bovine Tracheal Smooth Muscle Cells.
The BTSM cells were isolated as described elsewhere (Dekkers et al., 2007). Briefly, the BTSM tissue was chopped, and the tissue fragments were washed in Medium Plus [DMEM supplemented with sodium pyruvate (1 mM), nonessential amino-acid mixture (1:100), gentamicin (45 µg/ml), penicillin (100 U/ml), streptomycin (100 µg/ml), amphotericin B (1.5 µg/ml), and fetal bovine serum (FBS, 0.5%)]. Enzymatic digestion was performed in Medium Plus, supplemented with collagenase P (0.75 mg/ml), papain (1 mg/ml), and soybean trypsin inhibitor (1 mg/ml). The suspension was incubated in an incubator shaker (Innova 4000) at 37°C, 55 rpm for 20 minutes, followed by a 10-minute period of shaking at 70 rpm. After filtration of the obtained suspension over a 50 μm gauze, the cells were washed in Medium Plus, supplemented with 10% FBS. For all protocols, cells were used in passages 1–2.
Transfection of BTSM Cells with Green Fluorescent Protein Expression Vectors.
The BTSM cells were plated at a density of 30,000 cells/well in 24-well culture plates and allowed to attach overnight, or grown to 95% confluency in 100 mm culture dishes. Subsequently, cells were washed twice with phosphate-buffered saline (PBS). Transfections in 24-well culture plates were performed using a mixture of 2 μl lipofectamine 2000 and 0.1 µg expression vector [green fluorescent protein (GFP) or GFP-FAK] or 0.8 µg expression vector (GFP, GFP-FAT, or GFP-FRNK) for 6 hours in 120 µl plain DMEM without serum and antibiotics. For transfections in the 100 mm dishes, a mixture of 60 µl of lipofectamine 2000 and 3 µg or 24 µg of expression vector, respectively, in 3.6 ml of DMEM were used. After 6 hours, the cells were washed twice with PBS, and the medium was replaced by DMEM Zero supplemented with 0.1% FBS. Preliminary results indicated that transfection efficiency for GFP reached 30% ± 4% (n = 3).
Cell Adhesion Assay.
Collagen-coated (50 µg/ml) culture plates were prepared as described elsewhere (Dekkers et al., 2007). The method for measurement of cell adhesion was adapted from Orian-Rousseau et al. (1998). Untransfected, GFP-, GFP-FAK-, GFP-FAT-, or GFP-FRNK-transfected BTSM cells were harvested from 100 mm dishes by trypsinization. Cells were washed, resuspended in Medium Plus, and transferred into uncoated or collagen-coated 24-well culture plates at a density of 50,000 cells/well and placed back in the incubator. At varying time intervals, the plates were removed from the incubator, and the overlying medium was removed by gentle aspiration. After washing with 0.5 ml of PBS at 37°C, the cells were fixed with 70% ethanol for 15 minutes at 4°C. Subsequently, the plates were air dried for at least 30 minutes at 37°C and were stained for 25 minutes at room temperature using 0.1% crystal violet in water (0.3 ml/well). The cells were rinsed briefly with water and air dried. The stain was solubilized at room temperature using 10% acetic acid in water (0.5 ml/well) and quantified by colorimetric analysis (550 nm, Bio-Rad 680 plate reader; Bio-Rad Laboratories, Hercules, CA).
Western Blot Analysis.
For the measurement of the phosphorylation of FAK, the BTSM cells were cultured on uncoated or collagen I (50 µg/ml)–coated surfaces. At varying periods of time, the culture medium—also containing nonadhered cells—was removed gently, and the attached cells were lysed in homogenization buffer [composition in mM: Tris-HCl 50.0, NaCl 150.0, EDTA 1.0, PMSF 1.0, Na3VO4 1.0, NaF 1.0, pH 7.4, supplemented with leupeptin 10 µg/ml, aprotinin 10 µg/ml, pepstatin 10 µg/ml, Na-deoxycholate 0.25%, and Igepal 1% (NP-40)]. The protein content was determined, and equal amounts of protein were subjected to electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were subsequently blocked in blocking buffer [composition: Tris-HCl 50.0 mM, NaCl 150.0 mM, Tween-20 0.1%, and dried milk powder 5% (FAK) or bovine serum albumin (BSA) 5% (pFAK)] for 60 minutes at room temperature. Next, the membranes were incubated overnight at 4°C with primary antibodies (rabbit anti-FAK 1:2000 and rabbit anti-pFAK 1:1000, dilutions in blocking buffer containing BSA 5% or BSA 3%, respectively). After three washes with TBST (Tris-buffered saline/Tween 20 0.1% containing Tris-HCl 50.0 mM, NaCl 150.0 mM, and Tween 20 0.1%) of 10 minutes each, the membranes were incubated with horseradish peroxidase-labeled secondary anti-rabbit antibodies (dilution 1:2000 in blocking buffer containing 5% or 3% BSA, respectively) at room temperature for 90 minutes, followed by another three washes with TBST 0.1%. The antibodies were then visualized on film using enhanced chemiluminescence reagents and analyzed by densitometry (TotalLab, Newcastle upon Tyne, UK). Bands for pFAK were normalized to total FAK expression. Data are expressed as the percentage of the maximal FAK phosphorylation of matched samples run on the same gels.
Alamar Blue Proliferation Assay.
Alamar blue conversion was used to determine the changes in cell number. Previous studies have shown that changes in the conversion of Alamar blue closely match changes in absolute cell number (Dekkers et al., 2012). The BTSM cells were plated on uncoated or collagen I (1–100 µg/ml)–coated 24-well culture plates at a density of 30,000 cells/well and were allowed to attach overnight in Medium Plus, containing 10% FBS. The next day, the cells were washed twice with PBS and were made quiescent by incubation in Medium Zero, supplemented with 0.1% FBS for 3 days. Cells were then incubated with or without PDGF-AB (10 ng/ml) for 4 days in Medium Zero. Thereafter, the cells were washed two times with PBS and incubated with Hanks’ balanced salt solution containing 5% (v/v) Alamar blue solution. Conversion of Alamar blue into its reduced form by mitochondrial cytochromes was quantified by fluorometric analysis, as indicated by the manufacturer (Biosource, Camarillo, CA). Data were expressed as the percentage of Alamar blue conversion by unstimulated, vehicle-treated BTSM cells. When applied, PF-573228 (10–1000 nM), PP2 (10 μM), U0126 (3 μM), LY294002 (10 μM), or SB203580 (10 μM) were present during the entire incubation period. For overexpression of GFP, GFP-FAK, GFP-FAT, or GFP-FRNK, the BTSM cells were transfected with the vectors after attachment, and subsequently cells were made quiescent as previously described.
[3H]Thymidine-incorporation was performed as described elsewhere (Dekkers et al., 2007, 2009b). The BTSM cells were plated on uncoated or collagen I–coated 24-well culture plates at a density of 30,000 cells/well and were allowed to attach overnight in Medium Plus. The next day, the cells were transfected with the GFP, GFP-FAK, GFP-FAT, or GFP-FRNK, were washed with PBS, and were made quiescent by incubation in Medium Zero, supplemented with 0.1% FBS for 72 hours. Subsequently, the cells were washed and incubated in the absence or presence of PDGF (10 ng/ml) in Medium Zero for 28 hours, with the last 24 hours in the presence of [methyl-3H]thymidine (0.25 µCi/ml). After incubation, the cells were washed with PBS at room temperature. Subsequently, the cells were treated with ice-cold 5% trichloroacetic acid on ice for 30 minutes, and the acid-insoluble fraction was dissolved in NaOH (1 M). Incorporated [3H]thymidine was quantified by liquid-scintillation counting using a Beckman LS1701 β-counter (Beckman Coulter, Brea, CA).
DMEM, FBS, sodium pyruvate solution, nonessential amino acid mixture, gentamicin solution, penicillin/streptomycin solution, and amphotericin B solution (Fungizone) were obtained from Gibco BRL Life Technologies (Paisley, UK). Bovine serum albumin, apo-transferrin (human), leupeptin, aprotinin, pepstatin, soybean trypsin inhibitor, insulin (bovine pancreas) and (−)-isoproterenol hydrochloride were obtained from Sigma-Aldrich. PDGF-AB (human) was from Bachem (Weil am Rhein, Germany). Methacholine was obtained from ICN Biomedicals (Costa Mesa, CA). Anti-FAK was from Cell Signaling (Boston, MA). Anti-FAK [pY397] and Alamar blue were from Biosource. Collagenase P and papain were from Boehringer (Mannheim, Germany). Monomeric collagen type I (calf skin) was from Fluka (Buchs, Switzerland). Lipofectamine was from Invitrogen (Paisley, UK). PF-573228 and L(+)-ascorbic acid were from Merck (Darmstadt, Germany). SB203580, LY294002, U0126, and PP2 were obtained from Tocris Cookson (Bristol, UK). The pEGFP expression plasmids (Clontech) encoding FAK, and the FAK deletion mutants FAT and FRNK coupled to GFP were kindly provided by Dr. B. van de Water and Dr. S. E. Le Dévédec from the Division of Toxicology, Leiden Amsterdam Center for Drug Research (Ilic et al., 1998; Van de Water et al., 2001). All used chemicals were of analytic grade.
Data represent the mean ± S.E.M., from n separate experiments. Statistically significant differences between the means were calculated by use of one-way analysis of variance for repeated measurements, followed by a Student-Newman-Keuls multiple comparisons test. P < 0.05 was considered statistically significant.
Role of Focal Adhesion Kinase in Bovine Tracheal Smooth Muscle Cell Adhesion.
To investigate the effects of collagen I on ASM cell adhesion, we removed the BTSM cells from the culture dish by trypsinization and replated them onto uncoated plastic or monomeric collagen I (50 μg/ml)–coated culture plates. The BTSM cells adhered to both substrates within 8 hours, without differences between plastic or collagen I (Fig. 1A). To assess the changes in FAK activation during adhesion, we plated the BTSM cells, and after 1, 2, 4, 6, and 24 hours the nonadhered cells were removed, the adhered cells were lysed, and the FAK phosphorylation was determined. A statistically significant increase (P < 0.05) in FAK phosphorylation was observed in the cells adhering to the plastic and in collagen I compared with the cells in suspension (Fig. 1B). No differences in FAK phosphorylation were observed between either substrate. No FAK phosphorylation was observed in the nonadhered cells (unpublished data).
To investigate the role of FAK in ASM cell adhesion, we transfected the BTSM cells with GFP expression vectors encoding GFP (control), GFP-FAK, or the FAK deletion mutants GFP-FAT and GFP-FRNK. In the successfully transfected cells, the expression of GFP-FAK, GFP-FAT, and GFP-FRNK was detected in the focal adhesion sites (Fig. 2), which corresponds with previous findings in rabbit primary synovial fibroblasts (Ilic et al., 1998). By contrast, expression of GFP was observed diffusely throughout the cytoplasm. The cells expressing GFP, GFP-FAT, and GFP-FRNK remained elongated, whereas the cells overexpressing GFP-FAK lost their elongated appearance. To assess whether FAK activation was required for ASM cell adhesion, we transfected the BTSM cells with the expression vectors, then trypsinized them and replated them onto plastic. No effects of overexpression of GFP-FAK on cell adhesion were observed, whereas overexpression of GFP-FRNK or GFP-FAT statistically significantly reduced the cell adhesion (Fig. 3). Cell adhesion was maximally reduced at t = 24 hours, reaching 62% ± 8% (P < 0.001) in GFP-FRNK transfected cells and 67% ± 12% (P < 0.01) in GFP-FAT transfected cells compared with the GFP transfected cells. Of note, the inhibition of BTSM cell adhesion was comparable to the transfection efficiency (30% ± 4%). Collectively, these results indicate that endogenously expressed FAK is sufficient for BTSM cell adhesion, which can be inhibited by overexpression of GFP-FAT or GFP-FRNK.
Role of Focal Adhesion Kinase in Collagen I–Induced Bovine Tracheal Smooth Muscle Phenotype Switching.
Culturing of BTSM cells on collagen I increased the cell number in a concentration-dependent manner (Fig. 4A). The concentration of collagen required for a 50% increase (EC50) in the cell number was 14.1 ± 1.8 μg/ml. The collagen I–induced increase in BTSM proliferation was also time dependent, reaching 153% ± 12% at day 4 (P < 0.001; Fig. 4B). To assess whether the increases in cell number were associated with FAK activation, we cultured the BTSM cells on uncoated plastic or collagen I (50 μg/ml); the cells were lysed after 1, 2, 3, or 4 days of culture and analyzed for FAK phosphorylation. Culturing on collagen I increased FAK phosphorylation at days 2, 3, and 4 (P < 0.05; Fig. 4C). No statistically significant changes in total FAK expression were observed during the treatment period. In agreement with the findings for FAK phosphorylation during cell adhesion, no statistically significant increase in phosphorylation of FAK was observed on a collagen I matrix after 1 day.
To investigate the role of FAK in BTSM cell proliferation, we plated the cells on plastic or collagen I (50 µg/ml), allowed them to attach overnight, and transfected them with GFP, GFP-FAK, GFP-FAT, or GFP-FRNK expression vectors, and the cells were subsequently made quiescent and then stimulated with vehicle or PDGF-AB (10 ng/ml) for 4 days. As previous studies (Hirst et al., 2000a; Nguyen et al., 2005; Dekkers et al., 2010) have shown, collagen I enhanced the growth factor–induced proliferation. Although the value was not statistically significant, overexpression of GFP-FAK tended to decrease the proliferation induced by both PDGF and collagen I (Fig. 5A). Moreover, no effects of GFP-FAK overexpression were observed on the DNA synthesis induced by PDGF, collagen I, or both in combination (Fig. 5B). Overexpression of GFP-FAT or GFP-FRNK fully inhibited the increase in BTSM cell number induced by collagen I, whereas no significant effects of the inhibitory proteins were observed on PDGF-induced proliferation (Fig. 5C). PDGF-induced proliferation on collagen I was normalized to the level observed for PDGF-induced proliferation in cells cultured on plastic. No significant effects of overexpression of GFP-FAT or GFP-FRNK were observed on cell number in the absence of collagen I or PDGF. Similar effects were observed for GFP-FAT and GFP-FRNK on collagen I–induced DNA synthesis (Fig. 5D). Fully in line with these findings, collagen I–induced BTSM cell proliferation was also inhibited in a concentration-dependent manner by the specific pharmacologic FAK inhibitor PF-573228 (FAK inhibitor II) at concentrations that were specific for FAK inhibition (IC50 = 65 ± 16 nM; Fig. 6A) (Slack-Davis et al., 2007).
To assess whether activation of FAK was also required for the induction of a hypocontractile phenotype by collagen I, we cultured the BTSM strips in the absence and presence of collagen I (50 μg/ml) and/or PF-573228 (100 nM) for 4 days. As described elsewhere (Dekkers et al., 2007, 2012), culturing the BTSM strips in the presence of collagen I for 4 days statistically significantly (P < 0.05) reduced the maximal methacholine-induced contractile force compared with the vehicle-treated control strips (Fig. 6B; Table 1). Combined treatment with PF-573228 prevented the induction of a hypocontractile phenotype by collagen I, whereas the inhibitor by itself did not affect BTSM contractility. The sensitivity (pEC50) for methacholine was unaffected by all treatments (Table 1).
Role of Src, MEK, PI3-Kinase, and p38 MAPK in the Induction of a Proliferative, Hypocontractile ASM Phenotype by Collagen I.
To determine the contribution of downstream signaling pathways of FAK in the induction of a proliferative, hypocontractile ASM phenotype by collagen I, we cultured BTSM cells on plastic or collagen I in the absence and presence of the specific pharmacologic inhibitors Src (PP2, 10 µM), MEK (U0126, 3 µM), PI3-kinase (LY294002, 10 µM), or p38 MAPK (SB203580, 10 µM). Collagen I–induced proliferation was inhibited by all the inhibitors investigated (Fig. 7A). To investigate whether these pathways were involved in collagen I–induced hypocontractility as well, we cultured the BTSM strips in the absence and presence of collagen I (50 μg/ml) and the inhibitors for 4 days. As observed for proliferation, the collagen I–induced hypocontractility was normalized by all the inhibitors investigated (Fig. 7B; Table 2). The sensitivity for methacholine was unaffected by all treatments (Table 2).
In the current study, we demonstrate that the induction of a proliferative, hypocontractile ASM phenotype by monomeric collagen type I is dependent on the activation of FAK and downstream signaling pathways. Our results indicate that FAK is activated during BTSM cell adhesion and that overexpression of the two FAK deletion mutants FAT and FRNK, which compete with endogenous FAK for localization to the focal adhesions, inhibits cell adhesion. Moreover, FAK was activated during and required for collagen I–induced BTSM cell proliferation and phenotype switching. Pharmacologic inhibition of Src, MEK, PI3-kinase, and p38 MAPK signaling pathways, which may be activated downstream of FAK, inhibited the induction of a proliferative and hypocontractile phenotype induced by collagen I as well.
Airway hyperresponsiveness (AHR) is a characteristic feature of asthma and is defined by an exaggerated airway narrowing in response to either direct (histamine, methacholine) or indirect (exercise, cold air, hyperventilation) stimuli (Postma and Kerstjens, 1998). Variable AHR is observed after allergen exposure and is considered to reflect airway inflammation, whereas persistent AHR is considered to relate to structural changes in the airway wall, collectively termed airway remodeling (Cockcroft and Davis, 2006; Meurs et al., 2008). Increased ASM mass, as a feature of airway remodeling, is considered to be the most important factor contributing to AHR and decline in lung function in asthmatics (Ebina et al., 1993; Lambert et al., 1993; Oliver et al., 2007). Previously, we and others have shown that changes in the ECM environment surrounding the ASM may contribute to ASM accumulation (Hirst et al., 2000a; Johnson et al., 2004; Dekkers et al., 2007, 2010). Thus, culturing of ASM cells on collagen I matrices increased the proliferative responses, which were associated with a decreased contractile function of intact ASM tissue by this ECM protein, indicating that collagen I modulates the ASM phenotype from a contractile to a proliferative, hypocontractile phenotype (Hirst et al., 2000a; Dekkers et al., 2007, 2010, 2012). Little is known, however, about the signaling pathways involved in this process. In the current study, we found that culturing BTSM cells on collagen I increased the phosphorylation of FAK (a cytoplasmic protein tyrosine kinase activated by most integrins) in a time-dependent manner (Giancotti and Ruoslahti, 1999). Activation of FAK was found to be essential in collagen I–induced BTSM cell proliferation as overexpression of FAT and FRNK. FAT and FRNK inhibit FAK translocation and activation. Inhibition of one of these processes (or both) leads to the inhibition of the collagen-induced effects (Hildebrand et al., 1993; Richardson and Parsons, 1996). Accordingly, collagen I–induced proliferation was also inhibited by the specific pharmacologic inhibitor PF-573228 (FAK inhibitor II) at concentrations that have previously been found to be specific for FAK (Slack-Davis et al., 2007). In addition, this inhibitor fully reversed collagen I–induced hypocontractility, suggesting a key role of FAK in collagen I–induced ASM phenotype switching. Activation of FAK was also observed during the adhesion of BTSM cells to uncoated plastic and collagen I matrices, without differences between the two matrices. In addition, FAK activation was also required for BTSM cell adhesion, as indicated by its inhibition in FAT and FRNK overexpressing cells. The effects of overexpression of FAT and FRNK on collagen I–induced changes in BTSM cell number, as mentioned earlier, are unlikely to be due to changes in cell adhesion, as overexpression of these proteins only inhibited the collagen I–induced proliferative responses whereas no effects were observed on basal and PDGF-induced increases in cell number. In addition, no effects of overexpression of FAK itself were observed on the parameters assessed, suggesting that the endogenous expression of this kinase is sufficient and not the limiting factor in the activation of downstream signaling pathways.
Changes in FAK activation during the proliferative phase only became apparent after 2 days of culturing on collagen I, suggesting that FAK is not directly activated by collagen I–binding integrins but that additional processes may be required. Indeed, studies in vascular smooth muscle cells have indicated that culturing on monomeric collagen type I increased the expression of other ECM proteins, including fibronectin (Ichii et al., 2001), suggesting that the activation of FAK could be due to autocrine ECM deposition. This notion is also supported by previous findings showing that collagen I–induced increases in basal and growth factor–induced ASM proliferation required not only the collagen-binding integrin α2β1, but also the fibronectin-binding integrins α4β1 and α5β1 (Nguyen et al., 2005; Dekkers et al., 2010).
No effects of FAT or FRNK were observed on PDGF-induced proliferation, although in fibroblasts the activation of the PDGF receptor has been shown to induce FAK phosphorylation (Sieg et al., 2000). Also in BTSM cells, PDGF increased FAK phosphorylation in a time-dependent manner (unpublished data). The lack of effect of the deletion mutants, however, may be explained by the fact that activation of FAK by PDGF requires interaction of the receptor with the FERM domain, which is localized at the N terminus of the kinase (Sieg et al., 2000; Cox et al., 2006). Both deletion mutants, however, are derived from the C-terminal domain and inhibit FAK localization to the focal adhesions, which is required for FAK activation by integrins (Hildebrand et al., 1993; Richardson and Parsons, 1996), but do interfere with the activation of FAK via the FERM domain, providing a potential explanation for the lack of effect on PDGF-induced proliferation.
Phosphorylation of FAK at Tyr397 generates a high-affinity binding site for Src, which in turn fully activates FAK by phosphorylating Tyr576 and Tyr577 in the kinase domain (Hynes, 2002; Cox et al., 2006). Previous studies have found a critical role for Src in growth factor–induced ASM proliferation (Krymskaya et al., 2005). The essential role of Src in collagen I–induced BTSM phenotype modulation was indicated by the full inhibition of collagen I–induced proliferation and hypocontractility by the pharmacologic inhibitor PP2. Upon full activation by Src, FAK initiates a number of other signaling pathways, including the PI3-kinase and MAPK signaling pathways (Giancotti and Ruoslahti, 1999). Activation of these pathways has been found to be important in the response of ASM cells to growth factors. PI3-kinase activation has been associated with transcriptional activation and protein synthesis leading to ASM cell proliferation, hypertrophy, and both hypo- and hypercontractility (Walker et al., 1998; Halayko et al., 2004; Schaafsma et al., 2007; Dekkers et al., 2009b). Integrins may not only activate PI3-kinase through FAK but also via integrin-linked kinase (ILK), another cytoplasmic protein tyrosine kinase, which is activated by the β1 subunit of integrins (Liu et al., 2000). ILK has also been shown to be important in the regulation of contractile protein expression by human ASM cells. Knock down of ILK increased mRNA and protein expression of smooth muscle–specific myosin heavy chain (sm-MHC) via regulation of Akt, which is downstream of PI3-kinase (Wu et al., 2008). In our present study, inhibition of both PI3-kinase and FAK prevented collagen I–induced proliferation and hypocontractility, indicating the involvement of the both pathways in collagen I–induced BTSM proliferation. p42/p44 MAPK transfers growth-promoting signals to the nucleus and subsequently increase ASM proliferation (Gosens et al., 2008). In addition, p38 MAPK is involved in the regulation of growth factor-induced proliferation in ASM as well (Fernandes et al., 2004). Inhibition of the MAPK signaling pathways, either directly (p38 MAPK) or by inhibiting MEK, which is upstream of p42/p44 MAPK, also inhibited collagen I–induced BTSM proliferation and hypocontractility. Collectively, these findings suggest that collagen I–induced activation of FAK results in activation of Src and, subsequently, of PI3-kinase and MAPK signaling pathways downstream, which are all involved in collagen I–induced BTSM cell proliferation and hypocontractility.
In addition to its important role in ECM-induced phenotype switching, FAK has shown to be involved in acute ASM contractile responses. Thus, phosphorylation and membrane localization of the kinase are increased by mechanical strain and by contractile agonists (Smith et al., 1998; Tang et al., 1999; Gunst et al., 2003). Knockout of FAK in human tracheal smooth muscle strips decreases tension development, myosin light chain phosphorylation, and calcium signaling in response to the muscarinic receptor agonist acetylcholine and the membrane depolarizing stimulus KCl (Tang and Gunst, 2001), suggesting an important role of FAK in smooth muscle contraction. These findings and our current findings suggest that modulation of FAK activity in asthma may be an important new target in the treatment of ASM responsiveness and proliferation.
In conclusion, our study provides new insights on the signaling events leading to ASM phenotype modulation by collagen I. These signaling pathways involve activation of FAK as well as Src, MEK, PI3-kinase, and p38 MAPK downstream. Moreover, our results indicate that modulation of FAK activity may be a new target in the treatment of both variable and persistent AHR in asthmatics.
The authors thank Drs. B. van de Water and S. E. Le Dévédec from the Division of Toxicology, Leiden Amsterdam Center for Drug Research for the GFP, GFP-FAK, GFP-FAT, and GFP-FRNK expression plasmids.
Participated in research design: Dekkers, Spanjer, van der Schuyt, Kuik, Zaagsma, Meurs.
Conducted experiments: Dekkers, Spanjer, van der Schuyt, Kuik.
Performed data analysis: Dekkers, Spanjer, van der Schuyt, Kuik, Zaagsma, Meurs.
Wrote or contributed to the writing of the manuscript: Dekkers, Spanjer, van der Schuyt, Kuik, Zaagsma, Meurs.
- Received January 7, 2013.
- Accepted April 15, 2013.
This work was financially supported by the Netherlands Asthma Foundation [Grant 3.2.03.36].
- airway hyperresponsiveness
- airway smooth muscle
- bovine serum albumin
- bovine tracheal smooth muscle
- Dulbecco’s modified Eagle’s medium
- extracellular matrix
- focal adhesion kinase
- focal adhesion targeting
- fetal bovine serum
- FAK-related nonkinase
- green fluorescent protein
- integrin-linked kinase
- mitogen-activated protein kinase
- mitogen-activated protein kinase kinase
- platelet-derived growth factor
- 6-((4-((3-(methanesulfonyl)benzyl)amino)-5-trifluoromethylpyrimidin-2-yl) amino)-3,4-dihydro-1H-quinolin-2-one
- 1,4-diamino-2,3-dicyano-1,4-bis [2-aminophenylthio]butadiene
- Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics